N-terminal modification of RANTES and methods of use

ABSTRACT

N-terminally modified RANTES derivatives are disclosed. The derivatives effectively block the inflammatory effects of RANTES, and are useful for the treatment of asthma, allergic rhinitis, atopic dermatitis, atheroma/atherosclerosis, and rheumatoid arthritis. Additionally, the compounds are useful for the treatment of HIV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/678,851, filed Oct. 4, 2000 (now pending), which application is acontinuation of U.S. patent application Ser. No. 09/141,833, filed Aug.28, 1998 (now U.S. Pat. No. 6,168,784), which application claimspriority from U.S. Prov. Appl. No. 60/056,292 (filed Sep. 3, 1997), U.S.Prov. Appl. No. 60/077,874 (filed Mar. 13, 1998), and U.S. Prov. Appl.No. 60/090,834 (filed Jun. 26, 1998), all of which applications andpatents are hereby incorporated by reference herein in their entirety.

TECHNICAL FIELD

The invention relates to N-terminally modified RANTES derivatives thateffectively lock the inflammatory effects of RANTES, and are thus usefulfor the treatment of asthma, allergic rhinitis, atopic dermatitis,atheroma/atherosclerosis, and rheumatoid arthritis. Additionally, thecompounds are useful for the treatment of human immune deficiency virus(“HIV”).

BACKGROUND

The protein known as RANTES is a member of a large family of cytokinesknown as chemokines, and is classified as a .beta.-chemokine. It has asixty-eight amino acid sequence (SEQ ID NO:1). A receptor for RANTES hasrecently been cloned (Gao, et al., J. Exp. Med. 177:1421-7 (1993);Neote, et al., Cell 72:415-25 (1993)), which has been shown to bindchemokines in the order of potency of MIP-1.alpha.>RANTES.

Chemokines have the ability to recruit and activate a wide variety ofproinflammatory cell types, and RANTES has been shown to elicit aninflammatory response in vivo. RANTES, along with the natural ligandsfor the CCR5 chemokine receptor, MIP-1α, MIP-1β, were found to inhibithuman immune deficiency virus type-1 (“HIV-1”) infection (Cocchi, etal., Science 270:1811-1815 (1995)), leading to the identification ofCCR5 as the major co-receptor for primary isolates of HIV-1, HIV-2 andSIV-1 (Deng, et al., Nature 381:661-666 (1996); Doranz, et al., Cell85:1149-1158 (1996); Choe, et al., Cell 85:1135-1148 (1996); Chen, etal., J. Virol. 71:2705-2714 (1997); and Alkhatib, et al., Science272:1955-1958 (1996)). However, although RANTES consistently inhibitsHIV-1 replication in peripheral blood mononuclear cells, it does notblock infection of primary macrophage cultures, which suggests thatRANTES would not influence HIV replication in non-lymphocyte cell types.

N-terminal modifications of RANTES result in antagonists that can blockHIV-1 infection without signaling calcium flux (Mack, et al., J. Exp.Med. 187:1215-1224 (1998) and Proudfoot, et al., J. Biol. Chem.271:2599-2603 (1996)). These modifications include N-terminal truncation[RANTES 9-68] (Arenzana-Seisdedos, et al., Nature 383:400 (1996)), andaddition of methionine (“Met-RANTES”) or aminooxypentane (“AOP-RANTES”)at the N-terminus of RANTES (Mack, et al., supra and Simmons, et al.,Science 276:276-279 (1997)). It has been reported that the Met-RANTESand AOP-RANTES derivatives are antagonists of RANTES. Further,N-terminally modified RANTES, with a higher affinity for CCR5 thannative RANTES are more potent than native RANTES in blocking infection(Simmons, et al., supra). Chemokine receptor antagonists that arepotent, selective, and achieve full receptor occupancy would clearly beuseful for the treatment of HIV-1 in infected individuals. Surprisingly,compounds have been discovered with this spectrum of activity. Thesederivatives inhibited infection of many different cell types, includingmacrophages and lymphocytes.

Additionally, antagonists of RANTES effectively block its inflammatoryeffects, and are thus useful for the treatment of asthma, allergicrhinitis, atopic dermatitis, viral diseases, atheroma/atheroschleosis,rheumatoid arthritis and organ transplant rejection.

Certain derivatives of RANTES are disclosed in Wells, et al.,International Application WO 96/17935.

SUMMARY OF THE INVENTION

The polypeptides provided by the present invention are derivatives ofRANTES modified at the N-terminus; they are antagonists of RANTES,and/or of MIP-1α, and/or MIP-1β.

The polypeptides have the general formula: R¹-RANTES (2-68) (SEQ IDNO:2) where R¹ is CH₃—(CH₂)_(n)—X—; in which X is—C(O)—NH—CH₂—C(O)—,—NHCH₂ —C(O)—, —ONH—CH₂ —C(O)—, —OCH₂—CH₂—C(O)—,—CH═CH—C(O)—, —C(O)—, or a covalent bond; and n is an integer from 4-8.

Also provided are pharmaceutical compositions and methods of treatingdisease states, including HIV infection, by administeringtherapeutically effective amounts of compounds comprising RANTESderivatives, or pharmaceutically acceptable salts thereof.

The invention also provides for polypeptides of the formula R¹-RANTES(2-68) (SEQ ID NO:2) that have been modified by the grafting ofpolyethylene glycol (“PEG”) chains or PEG-based chains onto the RANTESportion of the polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chemical synthesis of n-nonyl-RANTES.

FIG. 2 shows the effect of AOP-RANTES treatment on HIV infection inhu-PBL-SCID mice.

FIG. 3 depicts the partial sequences of several HIV-1 clones.

FIG. 4 shows the in vitro effect of AOP-RANTES treatment on R5 viruses.

FIG. 5 depicts the effect of AOP-RANTES on HIV-1 infection in PBMCcultures.

FIG. 6 shows the effect of n-nonanoyl-RANTES (2-68) on HIV-1 infection.

FIG. 7 shows the inhibition of HIV-1 infection by AOP- and NNY-RANTES incultured primary human PBMC. Virus replication was measured by p24capsid antigen production after 5-7 days of infection. FIG. 7A showsinfection with the R5 SF162 isolate and FIG. 7B shows infection with twoR.5 variants of HIV-I 242.

FIG. 8 shows inhibition of HIV-1 infection by AOP- or NNY-RANTES inhu-PBL-SCID mice. CCR5 antagonists were delivered by subcutaneouslyimplanted osmotic pumps at the rate of 2.5 μg/hr beginning 1 day beforeHIV-1 infection. A single dose of 1 mg of AOP-RANTES (FIG. 8A) orNNY-RANTES (FIGS. 8B and 8C) was administered just prior to HIV-1challenge. Data presented are plasma HIV RNA copies/ml at 1-4 weeksafter infection, and each point represents the value for a singleanimal. The horizontal bar in each panel represents the duration ofcontinuous AOP- or NNY-RANTES administration.

DESCRIPTION OF SPECIFIC EMBODIMENTS

RANTES has the sequence: SPYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNPAVVFVTRKNR QVCANPEKKW VREYINSLEM S

The polypeptides of the present invention thus have the sequence:R¹-X-PYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKWVREYINSLEM Sor have a sequence which is substantially homologous with any of theabove sequences. Stated another way, the polypeptides of the inventionhave the general formula:R¹-RANTES (2-68)where R¹ is CH₃—(CH₂)_(n)—X—; in which X is —C(O)—NH—CH₂—C(O)—,—NHCH₂—C(O)—, —ONH—CH₂—C(O)—, —OCH₂—CH₂—C(O)—, —CH═CH—C(O)—, —C(O)—, ora covalent bond; and n is an integer of 4-8; or a pharmaceuticallyacceptable salt thereof.

The term “substantially homologous” when used herein includes amino acidsequences having at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%sequence homology with the given sequence (99% preference). This termcan include, but is not limited to, amino acid sequences having from 1to 20, from 1 to 10 or from 1 to 5 single amino acid deletions,insertions or substitutions relative to a given sequence provided thatthe resultant polypeptide acts as an antagonist to RANTES.

It should be noted that it is well known in the art that certain aminoacids can be replaced with others resulting in no substantial change inthe properties of a polypeptide, including but not limited toconservative substitutions of amino acids. Such possibilities are withinthe scope of the present invention. It should also be noted thatdeletions or insertions of amino acids can often be made which do notsubstantially change the properties of a polypeptide. The presentinvention includes such deletions or insertions (which may be, forexample up to 10, 20 or 50% of the length of the specific antagonistssequence given above).

The term “pharmaceutically acceptable salt” is used herein to mean asalt that retains the biological effectiveness and properties of thepolypeptides of the invention and which are not biologically orotherwise undesirable. Salts may be derived from acids or bases. Acidaddition salts are derived from inorganic acids, such as hydrochloricacid, hydrobromic acid, sulfuric acid (giving the sulfate and bisulfatesalts), nitric acid, phosphoric acid and the like, and organic acidssuch as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, salicylic acid,p-toluenesulfonic acid, and the like. Base addition salts may be derivedfrom inorganic bases, and include sodium, potassium, lithium, ammonium,calcium, magnesium salts, and the like. Salts derived from organic basesinclude those formed from primary, secondary and tertiary amines,substituted amines including naturally-occurring substituted amines, andcyclic amines, including isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, and the like. Preferred organic bases areisopropylamine, diethylamine, ethanolamine, piperidine, tromethamine,and choline.

It is well recognized that the properties of peptides can be enhanced bygrafting organic chain-like molecules onto them. These molecules areoften polyethylene glycol-based (“PEG”-based, i.e. based on therepeating unit —CH₂CH₂O—). See for example, Tsutsumi, et al, Jpn. J.Cancer Res. 85:9-12 (1994). PEG-based chains are amphiphilic,non-immunogenic and not susceptible to cleavage by proteolytic enzymes.Preparations of materials that have been modified by PEG or PEG-basedchains, have reduced immunogenicity and antigenicity. See for example,Abuchowski, et al, Journal of Biological Chemistry 252(11):3578-3581(1977). PEG also serves to increase the molecular size of the materialto which it is attached, thereby increasing its biological half-life.These beneficial properties of the PEG-modified materials make them veryuseful in a variety of therapeutic applications.

Accordingly, this invention also contemplates improving thepharmacokinetics of R¹-RANTES (2-68). This can be achieved by themodification or “PEGylation” of the polypeptides of the invention,R¹-RANTES (2-68), at sites that are likely to permit the proteins toretain their intrinsic biological activity. Such sites include, but notlimited to, the C-terminus of the polypeptide. The grafting of PEGchains or PEG-based chains onto proteins is known. See for example,Zalipsky, U.S. Pat. No. 5,122,614, which describes PEG that is convertedinto its N-succinimide carbonate derivative. Also known are PEG chainsmodified with reactive groups to facilitate grafting onto proteins. Seefor example, Harris, U.S. Pat. No. 5,739,208, which describes a PEGderivative that is activated with a sulfone moiety for selectiveattachment to thiol moieties on molecules and surfaces and Harris, etal., U.S. Pat. No. 5,672,662, which discloses active esters of PEG acidsthat have a single propionic or butanoic acid moiety. This area isextensively reviewed in Zalipsky, Bioconjugate Chemistry 6:150-165(1995). Besides use of PEG, Wright, EP 0 605 963 A2 describes linkingreagents that contain water soluble polymers that form a hydrazonelinkage with an aldehyde group on a protein. All of the aforementionedreferences are incorporated herein by reference.

As noted above, the polypeptides of the invention have the generalformula R¹-RANTES (2-68), where R¹ is CH₃—(CH₂)_(n)—X—; in which X isselected from the group consisting of —C(O)—NH—CH₂—C(O)—, —NHCH₂—C(O)—,—ONH—CH₂₋C(O)—, —OCH₂—CH₂—C(O)—, —CH═CH—C(O)—, —C(O)—, and a covalentbond; and n is an integer from 4-8. Polypeptides of particular interestinclude those listed below: Name n X n-hexanoyl-[Gly¹]RANTES (2-68) 4—C(O)—NH—CH₂—C(O)— n-nonanoyl-RANTES (2-68)^(a) 7 —C(O)—n-hexyl-[Gly¹]RANTES (2-68) 5 —NHCH₂—C(O)— n-nonyl-RANTES (2-68) 8covalent bond n-pentyloxy-[Gly¹]RANTES (2-68) 4 —ONH—CH₂—C(O)—3-pentyloxypropan-1-oyl-RANTES 4 —OCH₂—CH₂—C(O)— (2-68)non-2-en-1-ylRANTES (2-68) 5 —CH═CH—C(O)—^(a)n-nonanoyl-RANTES (2-68) is sometime referred to herein asNNY-RANTES

The polypeptides of the present invention are useful for blocking theeffects of RANTES⁻ and/or MIP-1α in mammals with respect to therecruitment and/or activation of pro-inflammatory cells. The presentinvention is therefore useful as an anti-inflammatory agent in thetreatment of diseases such as asthma, allergic rhinitis, atopicdermatitis, atheroma/atherosclerosis and rheumatoid arthritis.

M-tropic HIV viruses, also referred to as “R5” viruses, enter cells viathe CD4 and CCR5 receptors. T-tropic viruses, also referred to as “X4”viruses, enter cells via the CD4 and CXCR4 receptors. Dual-tropicviruses, also referred to as “R5x4” viruses, mediate entry via more thanone of the co-receptors. For example, HIV-1 242 is an M-tropic or R5virus. HIV-1 230 is a T-tropic or X4 virus. HIV-1 241 is a dual-tropicor R5X4 virus. Chesebro, et al., J. Virol. 70:9055-9059 (1996); Speck,et al., J. Virol. 71:7136-7139 (1997). For nomenclature classificationsof HIV-1 isolates by co-receptor, see Berger, et al., Nature 391:240(1997).

A preferred use of the polypeptides of the present invention is ininhibiting HIV-1 infection in mammals.

The term “treatment” as used herein covers any treatment of a disease ina mammal, particularly a human, and includes:

-   -   (i) preventing the disease from occurring in a subject which may        be predisposed to the disease but has not yet been diagnosed as        having it;    -   (ii) inhibiting the disease, i.e. arresting its development; or    -   (iii) relieving the disease, i.e. causing regression of the        disease.

The term “a disease state in mammals that is alleviated by treatmentwith a RANTES inhibitor” as used herein is intended to cover all diseasestates which are generally acknowledged in the art to be usefullytreated with RANTES inhibitors in general, and those disease stateswhich have been found to be usefully treated by the specific compoundsof our invention. These include, by way of illustration and notlimitation, asthma, allergic rhinitis, atopic dermatitis, viraldiseases, atheroma/atheroschleosis, rheumatoid arthritis and organtransplant rejection.

As used herein, the term “therapeutically effective amount” refers tothat amount of a polypeptide of the invention which, when administeredto a mammal in need thereof, is sufficient to effect treatment (asdefined above), for example, as an anti-inflammatory agent,anti-asthmatic agent, an immunosuppressive agent, or anti-autoimmunedisease agent to inhibit HIV-1 infection in mammals. The amount thatconstitutes a “therapeutically effective amount” will vary depending onthe polypeptide, the condition or disease and its severity, and themammal to be treated, its weight, age, etc., but may be determinedroutinely by one of ordinary skill in the art with regard tocontemporary knowledge and to this disclosure.

As used herein, the term “q.s.” means adding a quantity sufficient toachieve a stated function, e.g., to bring a solution to a desired volume(e.g., 100 mL).

Methods of Preparation

The polypeptides of the present invention are prepared by a combinationof chemical synthesis and chemical ligation techniques.

The synthesis of proteins by native chemical ligation is disclosed inInternational Patent Application PCT/US95/05668, InternationalPublication Number WO 96/34878, and a method of preparing proteinschemically modified at the N-terminal is disclosed in provisional PatentApplication 06/050,420, filed May 29, 1997, the disclosures of which areincorporated herein by reference. In general, a first oligopeptidecontaining a C-terminal thioester is reacted with a second oligopeptidewith an N-terminal cysteine having an unoxidized sulfhydryl side chain.The unoxidized sulfhydryl side chain of the N-terminal cysteine iscondensed with the C-terminal thioester in the presence of a catalyticamount of a thiol, preferably benzyl mercaptan, thiophenol,2-nitrothiophenol, 2-thiobenzoic acid, 2-thiopyridine, and the like. Anintermediate oligopeptide is produced by linking the first and secondoligopeptides via a β-aminothioester bond, which rearranges to producean oligopeptide product comprising the first and second oligopeptideslinked by an amide bond.

where P¹ is the first oligopeptide attached to a C-terminal thioester; Ris, for example benzyl; and P² is the second oligopeptide attached to anN-terminal cysteine having an unoxidized sulfhydryl side chain.

The polypeptides of the present invention are prepared as follows. Thefirst oligopeptide having a C-terminal thioester comprises the RANTESamino acid sequence 2-33 coupled to Gly at the 1-position, and isidentified as formula (4) below. This is prepared by solid phase peptidesynthesis on a thioester producing resin to give the compound of formula(2). The N-terminus of this material is then chemically modified, forexample by direct coupling of n-hexanoic acid to the resin to yield acompound of formula (3), followed by cleaving the product with hydrogenfluoride to yield the compound of formula (4). The chemically modifiedoligopeptide (4) is then purified, preferably by reverse-phasepreparative HPLC.

where PS is polymer support.

The second oligopeptide with an N-terminal cysteine having an unoxidizedsulfhydryl side chain comprises the RANTES amino acid sequence 34-68,and is identified as formula (5) below. It is prepared by solid phasepeptide synthesis on a Boc-Ser-OCH₂-Pam-resin, and is then cleaved withhydrogen fluoride. The oligopeptide with an N-terminal cysteine is thenpurified, preferably by reverse-phase preparative HPLC.

The first oligopeptide of formula (4) is then chemically ligated withthe second oligopeptide of formula (5) by the method disclosed above, toproduce RANTES chemically modified at the N-terminal, which is foldedand purified conventionally. Such polypeptides, would have the formula:

where X is —C(O)—NH—CH₂—C(O)—. Similarly, polypeptides of the inventionhaving other “X” groups such as —NHCH₂—C(O)—, —ONH—CH₂₋C(O)—,—OCH₂—CH₂—C(O)—, —CH═CH—C(O)—, —C(O)—, or a covalent bond, can besynthesized in a similar manner by using different materials to modifythe N-terminus.

The polypeptides may also be synthesized chemically, ribosomally in acell free system, or ribosomally within a cell.

Utility and Administration General Utility

The compounds of the present invention have been found to possessvaluable pharmacological properties, and have been shown to effectivelyblock the inflammatory effects of RANTES. Accordingly, they are usefulfor the treatment of asthma, allergic rhinitis, atopic dermatitis,atheroma/atheroschleosis, and rheumatoid arthritis. The compounds of thepresent invention have also been shown to inhibit HIV-1 infection invitro.

Testing

The potential of the compounds for utility against HIV-1 is determinedby the method, described in the following Examples. The potential of thecompounds for utility against inflammatory effects is determined bymethods well known to those skilled in the art.

General Administration

The polypeptides of this invention and their pharmaceutically acceptablesalts, i.e., the active ingredient, are administered at atherapeutically effective dosage, i.e., that amount which, whenadministered to a mammal in need thereof, is sufficient to effecttreatment, as described above. Administration of the polypeptidesdescribed herein can be via any of the accepted modes of administrationfor agents that serve similar utilities. As used herein, the terms“polypeptides of this invention”, “polypeptides”, “pharmaceuticallyacceptable salts of the polypeptides of the invention” and “activeingredient” are used interchangeably.

The level of the polypeptide in a formulation can vary within the fullrange employed by those skilled in the art, e.g., from about 0.01percent weight (% w) to about 99.99% w of the polypeptide based on thetotal formulation and about 0.01% w to 99.99% w excipient. Moretypically, the polypeptide will be present at a level of about 0.5% w toabout 80% w.

While human dosage levels have yet to be optimized for the polypeptidesof the invention, generally, a daily dose is from about 0.05 to 25 mgper kilogram body weight per day, and most preferably about 0.01 to 10mg per kilogram body weight per day. Thus, for administration to a 70 kgperson, the dosage range would be about 0.07 mg to 3.5 g per day,preferably about 3.5 mg to 1.75 g per day, and most preferably about 0.7mg to 0.7 g per day. The amount of polypeptide administered will, ofcourse, be dependent on the subject and disease state being treated, theseverity of the affliction, the manner and schedule of administrationand the judgment of the prescribing physician. Such use optimization iswell within the ambit of those of ordinary skill in the art.

Administration can be via any accepted systemic or local route, forexample, via parenteral, oral (particularly for infant formulations),intravenous, nasal, bronchial inhalation (i.e., aerosol formulation),transdermal or topical routes, in the form of solid, semi-solid orliquid or aerosol dosage forms, such as, for example, tablets, pills,capsules, powders, liquids, solutions, emulsion, injectables,suspensions, suppositories, aerosols or the like. The polypeptides ofthe invention can also be administered in sustained or controlledrelease dosage forms, including depot injections, osmotic pumps, pills,transdermal (including electrotransport) patches, and the like, for theprolonged administration of the polypeptide at a predetermined rate,preferably in unit dosage forms suitable for single administration ofprecise dosages. The compositions will include a conventionalpharmaceutical carrier or excipient and a polypeptide of the inventionand, in addition, may include other medicinal agents, pharmaceuticalagents, carriers, adjuvants, etc. Carriers can be selected from thevarious oils, including those of petroleum, animal, vegetable orsynthetic origin, for example, peanut oil, soybean oil, mineral oil,sesame oil, and the like. Water, saline, aqueous dextrose, and glycolsare preferred liquid carriers, particularly for injectable solutions.Suitable pharmaceutical carriers include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk, glycerol, propylene glycol, water, ethanol,and the like. Other suitable pharmaceutical carriers and theirformulations are described in “Remington's Pharmaceutical Sciences” byE. W. Martin.

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents and the like, such as forexample, sodium acetate, sorbitan monolaurate, triethanolamine oleate,etc.

Oral administration can be used to deliver the polypeptides of theinvention using a convenient daily dosage regimen which can be adjustedaccording to the degree of affliction. For such oral administration, apharmaceutically acceptable, non-toxic composition is formed by theincorporation of any of the normally employed excipients, such as, forexample, pharmaceutical grades of mannitol, lactose, starch, povidone,magnesium stearate, sodium saccharine, talcum, cellulose, croscarmellosesodium, glucose, gelatin, sucrose, magnesium carbonate, and the like.Such compositions take the form of solutions, suspensions, dispersibletablets, pills, capsules, powders, sustained release formulations andthe like.

The compositions may take the form of a capsule, pill or tablet and thusthe composition will contain, along with the active ingredient, adiluent such as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as croscarmellose sodium, starch or derivativesthereof; a lubricant such as magnesium stearate and the like; and abinder such as a starch, polyvinylpyrrolidone, gum acacia, gelatin,cellulose and derivatives thereof, and the like.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. a polypeptide of the invention(about 0.5% to about 20%) and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, suspending agents, emulsifyingagents, or solubilizing agents, pH buffering agents and the like, forexample, sodium acetate, sodium citrate, cyclodextrine derivatives,polyoxyethylene, sorbitan monolaurate or stearate, etc. Actual methodsof preparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. The composition orformulation to be administered will, in any event, contain a quantity ofthe active ingredient in an amount effective to alleviate the symptomsof the subject being treated. For oral administration to infants, aliquid formulation (such as a syrup or suspension) is preferred.

For a solid dosage form containing liquid, the solution or suspension,in for example propylene carbonate, vegetable oils or triglycerides, ispreferably encapsulated in a gelatin capsule. For a liquid dosage form,the solution, e.g. in a polyethylene glycol, may be diluted with asufficient quantity of a pharmaceutically acceptable liquid carrier,e.g. water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active ingredient in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g. propylenecarbonate) and the like, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells.

In applying the polypeptides of this invention to treatment of the aboveconditions, administration of the active ingredients described hereinare preferably administered parenterally. Parenteral administration isgenerally characterized by injection, either subcutaneously,intramuscularly or intravenously, and can include intradermal orintraperitoneal injections as well as intrasternal injection or infusiontechniques. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,solubility enhancers, and the like, such as for example, sodium acetate,polyoxyethylene, sorbitan monolaurate, triethanolamine oleate,cyclodextrins, etc.

The polypeptides of the present invention can be administeredparenterally, for example, by dissolving the polypeptide in a suitablesolvent (such as water or saline) or incorporation in a liposomalformulation followed, by dispersal into an acceptable infusion fluid. Atypical daily dose of a polypeptide of the invention can be administeredby one infusion, or by a series of infusions spaced over periodicintervals. For parenteral administration there are especially suitableaqueous solutions of an active ingredient in water-soluble form, forexample in the form of a water-soluble salt, or aqueous injectionsuspensions that contain viscosity-increasing substances, for examplesodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired,stabilizers. The active ingredient, optionally together with excipients,can also be in the form of a lyophilisate and can be made into asolution prior to parenteral administration by the addition of suitablesolvents.

A more recently devised approach for parenteral administration employsthe implantation of a slow-release or sustained-release system, suchthat a constant level of dosage is maintained. See, e.g., U.S. Pat. No.3,710,795, which is hereby incorporated by reference.

The percentage of the active ingredient contained in such parentalcompositions is highly dependent on the specific nature thereof, as wellas the activity of the polypeptide and the needs of the subject.However, percentages of active ingredient of 0.01% to 10% in solutionare employable, and will be higher if the composition is a solid whichwill be subsequently diluted to the above percentages. Preferably thecomposition will comprise 0.02-8% of the active ingredient in solution.

Another method of administering the polypeptides of the inventionutilizes both a bolus injection and a continuous infusion. This is aparticularly preferred method when the therapeutic treatment is for theprevention of HIV-1 infection.

Aerosol administration is an effective means for delivering thepolypeptides of the invention directly to the respiratory tract. Some ofthe advantages of this method are: 1) it circumvents the effects ofenzymatic degradation, poor absorption from the gastrointestinal tract,or loss of the therapeutic agent due to the hepatic first-pass effect;2) it administers active ingredients which would otherwise fail to reachtheir target sites in the respiratory tract due to their molecular size,charge or affinity to extra-pulmonary sites; 3) it provides for fastabsorption into the body via the alveoli of the lungs; and 4) it avoidsexposing other organ systems to the active ingredient, which isimportant where exposure might cause undesirable side effects. For thesereasons, aerosol administration is particularly advantageous fortreatment of asthma, local infections of the lung, and other diseases ordisease conditions of the lung and respiratory tract.

There are three types of pharmaceutical inhalation devices, nebulizersinhalers, metered-dose inhalers and dry powder inhalers. Nebulizerdevices produce a stream of high velocity air that causes thepolypeptide (which has been formulated in a liquid form) to spray as amist which is carried into the patient's respiratory tract. Metered-doseinhalers typically have the formulation packaged with a compressed gasand, upon actuation, discharge a measured amount of the polypeptide bycompressed gas, thus affording a reliable method of administering a setamount of agent. Dry powder inhalers administer the polypeptide in theform of a free flowing powder that can be dispersed in the patient'sair-stream during breathing by the device. In order to achieve a freeflowing powder, the polypeptide is formulated with an excipient, such aslactose. A measured amount of the polypeptide is stored in a capsuleform and is dispensed to the patient with each actuation. All of theabove methods can be used for administering the present invention.

Pharmaceutical formulations based on liposomes are also suitable for usewith the polypeptides of this invention. The benefits of liposomes arebelieved to be related to favorable changes in tissue distribution andpharmacokinetic parameters that result from liposome entrapment ofdrugs, and may be applied to the polypeptides of the present inventionby those skilled in the art. Controlled release liposomal liquidpharmaceutical formulations for injection or oral administration canalso be used.

For systemic administration via suppository, traditional binders andcarriers include, for example, polyethylene glycols or triglycerides,for example PEG 1000 (96%) and PEG 4000 (4%). Such suppositories may beformed from mixtures containing the active ingredient in the range offrom about 0.5 w/w % to about 10 w/w %; preferably from about 1 w/w % toabout 2 w/w %.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

Example 1 Preparation of n-Hexanoyl-[Gly¹]RANTES (2-33)

[Gly¹]RANTES (2-33) was synthesized by solid phase peptide synthesis ona thioester producing resin, and then chemically modified at theN-terminus by direct coupling of n-hexanoic acid to the resin. Theproduct was cleaved with hydrogen fluoride and purified by reverse-phasechromatography, to yield n-hexanoyl-[Gly¹]RANTES (2-33), a compound offormula (4).

Example 2 Preparation of RANTES (34-68)

RANTES (34-68) was synthesized by conventional solid phase peptidesynthesis on a Boc-Ser-OCH₂-Pam-resin followed by cleaving with hydrogenfluoride. The product was purified by reverse-phase preparative HPLC, toyield RANTES (34-68) or Cys³⁴-RANTES (35-68)-COOH, a compound of formula(5). Observed mass=4096.73 Da; Calculated mass=4097.8 Da (average).

Example 3 Preparation of n-Hexanoyl-[Gly¹]RANTES (2-68)

n-Hexanoyl-[Gly¹]RANTES (2-68) was prepared by chemically ligatingequimolar amounts of the compounds of formula (4) and (5) from Examples1 and 2 (22 mg each) in 6M guanidine hydrochloride, 200 mM of sodiumphosphate, 30 mM L-methionine, at pH 7 containing 1% v/v thiophenol.After 20 hours the reaction was complete, and the product was purifiedby reverse-phase semi-preparative HPLC and lyophilization, to yield 22.6mg of reduced n-hexanoyl-[Gly¹]RANTES (2-68).

Folding and formation of the disulfides was carried out in 8 mMcysteine, 1 mM cystine, 2M guanidine hydrochloride, 100 mM TRIS pH 8.00at a protein concentration of 1 mg/ml. Gentle stirring at roomtemperature resulted in quantitative folding, and purification byreverse-phase semi-preparative HPLC and lyophilization yielded 12.6 mgof folded n-hexanoyl-[Gly¹]RANTES (2-68). Observed mass=7915.09 Da;Calculated mass=7915.2 Da (average).

Example 4 Preparation of n-Nonanoyl-RANTES (2-68)

The peptide, n-nonanoyl-RANTES (2-68) was synthesized using the in situneutralization/HBTU (Spectrum Quality Products, Inc., Gardena, Calif.)activation protocols for Boc chemistry solid phase peptide synthesis asdescribed in Schnölzer, et al., Int. J. Pept. Protein Res. 40:180-193(1992). Boc-amino acids were purchased from Peptide Institute, Inc.(Osaka, Japan). Peptide α-carboxylates were assembled on aBoc-Ser-OCH₂-Pam resin (PE Applied Biosystems, Foster City, Calif.) andthe peptide α-thiocarboxylates on a thioester generating resin, asdescribed in Hojo, et al., Bull. Chem. Soc. Jpn. 64, 111-117 (1991). TheN-terminal modifications were incorporated by on-resin reaction ofRANTES (2-33) with the preformed n-nonanoic acid to given-nonanoyl-RANTES (2-33) thioester. Peptides were cleaved from the resinusing hydrogen fluoride containing 5% v/v p-cresol (FLUKA, Buehs,Switzerland) for 1 hour at 0° C. and purified by reversed-phase HPLC(VYDAC, Inc., Hesperia, Calif.) with a linear gradient of acetonitrileversus water containing 0.1% trifluoroacetic acid to yieldn-nonanoyl-RANTES (2-33)-S-Ac-Leu.

Native chemical ligation (Dawson, et al., Science 266:776-779 (1994)) ofthe fully unprotected peptide segment with RANTES (34-68) in aqueousbuffer gave the full length polypeptide in reduced form(n-nonanoyl-RANTES (2-68) reduced)), which was folded in aqueous bufferand purified by reversed-phase HPLC. The folded n-nonanoyl-RANTES (2-68)(8.4 mg) was homogeneous on HPLC. Observed mass=7899.96 Da; Calculatedmass=7900.21 Da (average).

Example 5 Preparation of n-Hexyl-[Gly¹]RANTES (2-68)

RANTES (2-33) was synthesized by solid phase peptide synthesis on athioester producing resin, and then chemically modified at theN-terminus by direct coupling of bromoacetic acid to the resin followedby n-hexylamine. The product was cleaved with hydrogen fluoride andpurified by reverse-phase chromatography, to yield n-hexyl-[Gly¹]RANTES(2-33).

This material was chemically ligated with a compound of formula (5) asshown in Example 3 above, to give n-hexyl-[Gly¹]RANTES (2-68) (5.4 mg).Observed mass=7901.36 Da; Calculated mass=7901.2 Da (average).

Example 6 Preparation of n-Nonyl-RANTES (2-68)

As shown in FIG. 1, RANTES (2-33) was synthesized by solid phase peptidesynthesis on a thioester producing resin, and then chemically modifiedat the N-terminus by direct coupling of 1-bromo-nonane to the resin. Theproduct was cleaved with hydrogen fluoride and purified by reverse-phasechromatography, to yield n-nonyl-RANTES (2-33)-S-Ac-Leu.

This material was chemically ligated with a compound of formula (5) asshown in Example 3 above, to give n-nonyl-RANTES (2-68) reduced, whichwas then folded and purified as shown in Example 3 above to give foldedn-nonyl-RANTES (2-68) (5.8 mg). Observed mass=7886.16 Da; Calculatedmass=7886.2 Da (average).

Example 7

By using the above methods the following compounds were similarlyprepared:

-   -   n-pentyloxy-[Gly¹]RANTES (2-68);    -   3-pentyloxypropan-1-oyl-RANTES (2-68); and    -   non-2-en-1-ylRANTES (2-68).

Example 8 Preparation of AOP-RANTES (2-68)

The peptides, aminooxypentane-RANTES (2-68) (“AOP-RANTES (2-68)”) wassynthesized using the in situ neutralization/HBTU (Spectrum QualityProducts, Inc., Gardena, Calif.) activation protocols for Boc chemistrysolid phase peptide synthesis as described in Schnölzer, et al., Int. J.Pept. Protein Res. 40:180-193 (1992). Boc-amino acids were purchasedfrom Peptide Institute, Inc. (Osaka, Japan). Peptide α-carboxylates wereassembled on a Boc-Ser-OCH₂-Pam resin (PE Applied Biosystems, FosterCity, Calif.) and the peptide α-thiocarboxylates on a thioestergenerating resin, as described in Hojo, et al., Bull. Chem. Soc. Jpn.64, 111-117 (1991). The N-terminal modifications were incorporated byon-resin reaction of RANTES (2-33) with the preformed oximen-pentyl-O—N═CHCOOH as the last step in the chain assembly to giveAOP-RANTES (2-33). Peptides were cleaved from the resin using hydrogenfluoride containing 5% v/v p-cresol (FLUKA, Buehs, Switzerland) for 1hour at 0° C. and purified by reversed-phase HPLC (VYDAC, Inc.,Hesperia, Calif.) with a linear gradient of acetonitrile versus watercontaining 0.1% trifluoroacetic acid. Native chemical ligation (Dawson,et al., Science 266:776-779 (1994)) of the fully unprotected peptidesegments AOP-RANTES (2-33) thioester with RANTES (34-68) in aqueousbuffer gave the full length polypeptide in reduced form, which wasfolded in aqueous buffer and purified by reversed-phase HPLC. The foldedAOP-RANTES was homogeneous on HPLC and gave a molecular mass of7901.2±0.8 Daltons (“Da”) on electrospray ionization mass spectroscopy(calculated average isotope composition 7901.2 Da

Example 9 Chemotaxis Assays

Human peripheral blood leukocytes are isolated from normal donorsaccording to established protocols for purification of monocytes, Tlymphocytes and neutrophils. A panel of CC and CXC chemokinereceptor-expressing test cells is constructed and evaluated followingexposure to serial dilutions of individual compounds of the presentinvention. Synthetic native RANTES, a native CXC-chemokine (“SDF-1α”),MPAV and MPBV are used as controls. The panel of cells represent humankidney embryonic epithelial (“HEK”) 293 cells transfected withexpression cassettes encoding various chemokine receptors includingCXCR4/Fusion/LESTR, CCR3, CCR5, CXC4 (these cells are available fromvarious commercial and/or academic sources or can be prepared followingstandard protocols). Leukocyte migration relative to the transfected HEK293 cells is evaluated using a 48-well microchamber, migration of thereceptor transfected HEK 293 cells also is assessed by the 48-wellmicrochamber technique with the polycarbonate filters (10 μm pore-size)precoated with Collagen type I (Collaborative Biomedical Products,Bedford, Mass.) (Neote, et al., supra; Risau, et al., Nature 387:671-674(1997); Angiololo, et al., Annals NY Acad. Sci. 795:158-167 (1996);Friedlander, et al., Science 870:1500-1502 (1995)). The results areexpressed as the chemotaxis index (“CI”) representing the fold increasein the cell migration induced by stimuli versus control medium. Allexperiments are performed at least two times. The statisticalsignificance of the difference between migration in response to stimuliand control are accessed by Student's T test.

Example 10 Receptor Binding Assays

Receptor binding assays are performed using a single concentration of¹²⁵I labeled chemokines in the presence of increasing concentrations ofunlabeled ligands following standard protocols. The binding data areanalyzed, for example, with a computer program such as LIGAND (P.Munson, Division of Computer Research and Technology, NIH, Bethesda,Md.). The binding data are subjected to Scatchard plots analysis withboth “one site” and “two site” models compared to native leukocytes orthe panel of receptor-transfected HEK 293 cells expressing CXCR4, CCR3,CCR5 or CXC4. The rate of competition for binding by unlabeled ligandsis calculated with the following formula: % inhibition=1−(Binding in thepresence of unlabeled chemokine/binding in the presence of mediumalone)×100.

Example 11 HIV-1 Inhibition Assays

A. Chemokine receptors act as co-factors for HIV-1 entry into CD4⁺cells. The CC chemokines MIP-1α, MIP-1β, RANTES and eotaxin can suppresssome strains of HIV replication in primary peripheral blood mononuclearcells (“PBMCs”) and chemokine receptor transfected cell lines. The viralproduced chemokine vMIP-1 inhibits some primary non-syncytium inducing(NSI) HIV strains when co-transfected with the NSI strain HIV-1co-receptor CCR5. CCR3 is the predominant chemokine receptor throughwhich eotaxin, RANTES and other CC chemokines activate eosinophils.RANTES and MIP-1α also can utilize the CCR1 receptor that is expressedon eosinophils. In addition, synthetic N-terminal variants of CC (e.g.Met-RANTES) and CXC (e.g. IL-8) chemokines function as receptorantagonists on eosinophils and neutrophils, whereas the nativestructures do not. Similarly, the CXC chemokine SDF-1α is a potentchemoattractant for leukocytes through activation of the receptorCXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of HIV-1.CXCR4 mediated HIV-1 fusion can be inhibited in some cells by SDF-1α.Thus, despite the sequence similarities between certain chemokines ofthe same family, the binding and antagonist/agonist properties for HIVinfection vary significantly.

Polypeptides of the invention are screened for receptor usage,inhibition of HIV infection, potency and breadth of activity against HIVinfection, induction of calcium mobilization and angiogenesis. Theassays are used to evaluate suppression of HIV-1 infection/replicationin U87/CD4 cells (a human glioma cell line) expressing HIV-1co-receptors and also in PBMCs.

The receptor-transfected U87/CD4 cells are obtainable by transfectingcells with an expression cassette encoding the respective receptorsfollowing standard protocols. The cells are maintained in Dulbecco'sMinimal Essential Medium containing 10% fetal calf serum (“FCS”),glutamine, antibiotics, 1 μg/ml puromycin (Sigma Chemicals) and 300μg/ml neomycin (G418; Sigma) and split twice a week. PBMCs are isolatedfrom healthy blood donors by Ficoll-Hypaque centrifugation, thenstimulated for 2-3 days with phytohemagglutinin (“PHA”) (5 μg/ml) andIL-2 (100 U/ml) (Simmons, et al., J. Virol. 70:8355-8360 (1996)). CD4⁺T-cells are purified from the activated PBMCs by positive selectionusing anti-CD4 immunomagnetic beads (DYNAL Inc.), screened for CCR-5defective alleles, and cells from allele defective or wild-type donorsused depending on the assay. HIV isolates are obtainable from varioussources including the NIAID HIV-1 Antigenic Variation study, or fromsimilar programs organized by the US Department of Defense or the WorldHealth Organization. Phenotypes of test viruses are tested by theirability to form syncytia (“SI”) in MT-2 cells that are cultured in RPMI1640 medium containing 10% FCS, glutamine and antibiotics, and splittwice a week. Human CC-chemokines MIP-1α, MIP-1β and RANTES, andCXC-chemokines SDF-1α stocks are compared for purity and potency.

B. Assay for Inhibition of HIV Infection

Compounds of the present invention are tested against a panel of U87/CD4cells stably expressing either CCR3, CCR5, CXC4 or CXCR4 receptorsexposed to HIV-1/NSI strains SL-2 and SF162 (macrophage-tropic strainsthat utilize the RANTES, MIP-1α and MIP-1β receptor CCR5 to gain entryinto CD4⁺ cells) and the dual-tropic syncytium inducing strains 89.6 and2028 (syncytium inducing dual tropic strains that can use CXCR4 and CCR3in addition to CCR5 for entry). Lymphocytes and CD4⁺ T-cells from donorsalso are tested. Serial concentrations ranging from 0 to 500 nM of thecross-over proteins are used. RANTES, MPBA, MPBV and SDF-1α are used ascontrols. Inhibition of HIV infection is reported as a percentage ofinfection relative to modular protein and control concentrations.

Purified lymphocytes are stimulated with PHA (0.5 μg/ml) and culturedfor 2-3 days at 2×106/ml in medium containing IL-2 (Boeringer-Mannheim,20 U/ml) before being used in infection assays. Cells are pre-treatedwith appropriate concentrations of chemokines for 30 minutes at 37° C.Approximately 400-1000 tissue culture infectious doses (“TCIDs”) ofvirus are added. The cells are washed 4 times and resuspended in anappropriate volume of media containing IL-2 and relevant chemokine atthe appropriate concentration. Cells are fed every 3 days with freshmedium contain IL-2 and chemokine. From days 3 through 7 post-infection,the cultures are examined microscopically for syncytium formation andthe supernatant analyzed for p24 antigen production using an enzymelinked immunoabsorbent assay (“ELISA”) (McKnight, et al., Virology201:8-18(1994); and Mosier, et al., Science 260:689-692 (1993)).Inhibitory doses a calculated relative to the final concentration ofchemokine in the culture on day 0. Virus production in the absence ofchemokine is designated as 100%, and the ratios of p24 antigenproduction in chemokine-containing cultures calculated relative to thispercentage. The chemokine concentrations (pg/ml) causing 50% and 90%reduction in p24 antigen production are determined by linear regressionanalysis. If the appropriate degree of inhibition is not achieved at thehighest or lowest chemokine concentration, a value of >or< is recorded.

Virus infectivity on the receptor expressing U87/CD4 cells is assessedby focus-forming units (FFU) (Simmons, et al, Science276:276-279(1997)). The FFU for viruses using more than one co-receptoris assessed separately for each appropriate co-receptor expressingU87/CD4 cell type. Cells are seeded into 48 well trays at 1×104cells/well overnight. The cells are then pre-treated for 30 minutes at37° C. with appropriate concentrations of chemokine in 75 μl. 100 FFU ofeach virus in 75 μl is added and incubated for 3 hours at 37° C. Cellsare washed 3 times and 500 μl of medium containing the appropriatechemokine at the correct concentration is added. After 5 days the cellsare fixed for 10 minutes in cold acetone:methanol (1:1) and analyzed forp24 antigen production. Stand potency of cross-over chemokines againstHIV infection

The breadth and potency of the inhibitory actions of the compounds ofthe present invention are tested against native CC-chemokines (MIP-1α,MIP-1β and RANTES) for M-tropic primary isolates of HIV-1, and against anative CXC-chemokine (SDF-1α) for T-tropic isolates inmitogen-stimulated primary CD4⁺ T-cells. The compounds are evaluated fortheir potency and spectrum of agonistic activity against HIV-1 strainsrelative to the native CC- and CXC-chemokines to identify the mostactive inhibitor of HIV-1 replication and the best template fortherapeutic development. The properties and activities of M-Tropic andT-tropic primary HIV-1 isolates are recorded and compared to inhibitionof infection by exposure to the cross-over chemokines relative to theHIV isolate designation, genetic subtype, and phenotype determined byability of an isolate to form (“SI”) or not form (“NSI”) syncytia inMT-2 cells, the ability of an isolate to replicate efficiently inactivated CD4⁺ T-cells from individuals homozygous for either wild-typeor delta-32 CCR5 alleles, and the ability of an isolate to replicate inU87/CD4 cells stably expressing either CCR5 or CXCR4. The median ID₅₀and ID₉₀ values (ng/ml) are calculated for each sample. A value of >indicates that 50% or 90% inhibition is not achieved at a chemokineconcentration of the highest tested in any experiment. The means fromtwo independent experiments are compared. FACS analysis of CCR5 andCXCR4 receptor expression levels, and/or competitive inhibition assay ofthe compounds of the present invention and receptor down-regulation alsomay be tested following standard protocols (Wu, et al., J. Exp. Med.185:168-169(1997); and Trkola, et al., Nature 384:184-186(1996)).

Example 12 Assay for Measuring Changes in Intracellular CalciumConcentration ([Ca⁺])

Calcium mobilization is indicative of receptor binding. The compounds ofthe present invention are assayed for calcium mobilization in purifiedneutrophils and eosinophils following standard protocols (Jose, et al.,J. Exp. Med. 179:881-887 (1994)). Purified neutrophils or eosinophilsare incubated with fura-2 acetoxymethyl ester (1-2.5 μM), washed 3 timesin 10 mM phosphate buffered saline (“PBS”) (without Ca²⁺/Mg²⁺) and 0.1%bovine serum albumin (“BSA”) (200×g, 8 min), and finally resuspended at2×106 cells/ml in 10 mM PBS (without Ca²⁺/Mg²⁺), 0.25% BSA, 10 mM HEPES,and 10 mM glucose. Aliquots of cells are placed in quartz cuvettes andthe external Ca²⁺ concentration adjusted to 1 mM with CaCl₂ Changes influorescence are measured at 37° C. using a fluorescencespectrophotometer at excitation wavelengths 340 nm and 380 nm andemission wavelength 510 nm. [Ca²⁺] levels are calculated using the ratioof the two fluorescence readings and a K for Ca²⁺ at 37° C. of 224 nM.

Example 13 CAM Assay for Angiogenic Activity

Angiogenic activities of compounds of the present invention areevaluated by the chick chorioallantoic membrane (CAM) assay (Oikawa, etal., Cancer Lett. 59:57-66 (1991)). Native chemokines are used ascontrols. Fertilized Plymouth Rock Leghorn eggs are incubated at 37° C.in a humidified atmosphere (relative humidity, approx. 70%). Testsamples are dissolved in sterile distilled water or PBS. Sterilizedsample solution is mixed with an equal volume of autoclaved 2%methylcellulose. Additional controls are prepared with vehicle only (1%methylcellulose solution). 20 μl of the sample solution is dropped onparafilm and dried up. The methylcellulose disks are stripped off fromthe parafilm and placed on a CAM of a 10 or 11 day old chick embryo.After 3 days, the CAMs are observed by means of an Olympus stereoscope.A 20% fat emulsion (Intralipos 20%, Midori-Juji, Osaka, Japan) isinjected into the CAM to increase the contrast between blood andsurrounding tissues (Danesi, et al., Clin. Cancer Research3:265-272(1997)). The CAMs are photographed for evaluation of angiogenicresponse. Angiogenic responses are graded as negative, positive orunclear on the basis of infiltration of blood vessels into the area ofthe implanted methylcellulose disk by different observers.

Example 14 AOP-RANTES Inhibits HIV-1 Replication In Vivo and In Vitro

Primary macrophage-tropic isolates of HIV-1 (R5 viruses) use CCR5 as aco-receptor for virus entry into target cells. CCR5 binds RANTES,MIP-1α, and MIP-1β, and each of these chemokines can block virus entryinto T cells at relatively high concentrations. N-terminal modificationsof RANTES with higher affinity for CCR5 binding have been tested forinhibition of virus entry and replication in both T cells andmacrophages in vitro. In addition, in vivo tests of the efficacy ofthese antagonists have been performed in SCID mice repopulated withhuman peripheral blood mononuclear cells (the “hu-PBL-SCID” model,described in Mosier, et al., Nature (London) 335:256-259 (1988) andMosier, et al., Science 251:791-794 (1991 )). AOP-RANTES is an effectiveinhibitor of M-tropic R5 virus replication in vitro at nanomolarconcentration. The same compound substantially reduces virus levels whenadministered to hu-PBL-SCID mice just prior to virus infection andcontinuously for the ensuing week. Virus rebounds once treatment isstopped, but the recovered virus shows no mutations in the V3 region andhas not altered co-receptor usage. First generation chemokineantagonists thus show antiviral efficacy in an animal model, and thisantiviral effect may not select for viral variants with alteredco-receptor utilization.

AOP-RANTES Inhibits HIV-1 Replication in hu-PBL-SCID Mice

Hu-PBL-SCID mice were generated by injection of 20×10⁶ PBMCs from anEBV-seronegative donor on day-14 or -15, using methods described inMosier, Adv. Immunol. 63:79-125 (1996), Picchio, et al., J. Virol.71:7124-7127 (1997), and Picchio, et al., J. Virol. 72:2002-2009 (1998).The mice were divided into groups of four mice each. Group 1 receivedimplantation of an Alzet 2001 mini-osmotic pump (ALZA Pharmaceuticals,Palo Alto, Calif.) containing 2.5 mg/ml of AOP-RANTES (delivered at arate of 1 μl per hour for a minimum of 200 hours). Group 2 receivedimplantation of a similar Alzet pump containing 2.5 mg/ml of BSA. TheAlzet pumps were implanted subcutaneously on day-1 (14 days after theSCID mice were reconstituted). Thus, on day-1, delivery of AOP-RANTESbegan for mice in Group 1. On the following day (day 0), the Group 1mice were given a single bolus injection of 1 mg AOP-RANTES and theGroup 2 mice were given a single bolus injection of 1 mg BSA. One hourlater, all mice were challenged with 1000 TCIDs of the M-tropic 242HIV-1 isolate (Chesebro, et al., supra and Speck, et al., supra).

Virus infection was monitored by plasmid HIV RNA levels, which weredetermined using the Roche Amplicor HIV Monitor assay, a quantitativePCR determination with a limit of detection of 200 copies/ml. OneAOP-RANTES mouse died on day 6 of a surgical complication of pumpreplacement. Plasma samples were obtained from all mice on day 7 (7 daysafter infection with HIV-1) and plasma HIV-1 RNA copy number measured ondays 7 and 14 (after infection). Plasma RNA levels 200 are undetectablewith the Roche assay, so two mice (#1 and #2) shown as 200 copies/ml onday 7 after infection had undetectable viral RNA levels. The reductionin plasma virus RNA levels by AOP-RANTES at day 7 after infection werehighly significant (p<0.001).

Plasma AOP-RANTES levels on day 7 were determined with a human RANTESELISA kit (R&D). Plasma concentrations of AOP-RANTES in the range of 1-5ng/ml suggest that plasma clearance is more rapid than Alzet pumpdelivery rate.

At the final time point, mice from each group were sacrificed fordetermination of the numbers of human CD3, CD4, CD8, and CD45-positivecells in two sites, the peritoneal cavity site of cell injection, andlocal lymph nodes (“LN”) draining the peritoneal cavity. The frequencyof human cell types was determined by staining with fluorochrome-labeledantibodies followed by fixation and flow cytometric analysis, asdescribed in Picchio, et al., J. Virol. 72:2002-2009 (1998) and Picchio,et al., J. Virol. 71:7124-7127 (1997). The % CD4 T cells was measured 14days after infection. This is illustrated in FIG. 2.

After cessation of AOP-RANTES treatment, virus bounced back in all mice.Infection was delayed and CD4 T cells spared (at least temporarily) byAOP-RANTES treatment, but virus infection was not blocked.

AOP-RANTES has now been shown to be non-toxic and to significantly delayHIV-1 virus infection. Approximately 50 mg/kg of neutralizing-antibodyis required to block infection of the mice.

Example 15 In Vitro Assays to Determine Inhibitory Effects of AOP-RANTESon Different HIV-1 Viruses

In vitro experiments have concentrated on the inhibitory effects ofAOP-RANTES on dual-tropic viruses that can use other co-receptors inaddition to CCR5. These in vitro experiments involved use of a p24 virusreplication assay as described in Mosier, et al., supra. Briefly,culture media was sampled at day 7 post-infection with the relevantHIV-1 isolate, and p24 was measured in pg/ml via an ELISA assay. Theresults are shown in FIG. 4, which shows that AOP-RANTES treatment invitro inhibits R5 viruses but can enhance replication by R5X4 viruses ina CCR5-independent manner. Purified CD4 T cells were activated with PHA(and not IL-2) for 3 days prior to infection with HIV-1 242, 241 or thedual tropic 89.6 isolate. CD4 T cells were derived from either a normaldonor (wild type “wt”) or a donor homozygous for the 32 bp deletion inthe CCR5 gene and thus having no expression of CCR5 on the cell surface(“del32”) (as described in Picchio, et al., J. Virol. 71:7124-7127(1997)).

As shown in FIG. 4, three different HIV-1 viruses were used: 241, 242,and 89.6. 89.6 is a dual-tropic HIV-1 isolate that uses multiplechemokine receptors, CCR2, CCR3, CCR5 and CXCR4, and is designatedR2R3R5X4. Replication of HIV-1 242 in del32 CD4 T cells was enhanced byAOP-RANTES, but replication of HIV-1 89.6 in these T cells was unchangedby AOP-RANTES, as shown in FIG. 4.

All M-tropic viruses (R5 viruses) that use CCR5 only are efficientlyinhibited. The dual tropic virus 241 is enhanced by some concentrationsof AOP-RANTES when infection is measured on whole PBMCs, but isinhibited with purified CD4 T cells are the targets for infection. 241infection was also enhanced by AOP-RANTES on purified CD4 T cells fromCCR5 null (del32/del32) donors, suggesting some activity via otherchemokine receptors. The dual-tropic 89.6 isolate that uses CCR2b, CCR3,and CSCR4 as well as CCR5 was inhibited about 50% by AOP-RANTES. Primaryisolates of dual-tropic viruses from patients were inhibited between80-90%. As might be expected, it appears that each dual-tropic virusisolate uses CCR5 to a greater or lesser extent. Adding AOP-RANTES to amixture of cell types can elicit strange responses, and its ability totrigger other CC receptors needs to be defined.

Example 16 Low Concentrations of AOP-RANTES Can Enhance Replication ofDual-Tropic HIV-1 in PBMC Cultures from Selected Donors

Isolated PBMCs activated with PHA and IL-2 were infected with eitherHIV-1 242, 241 or 230 (see FIGS. 3 and 5) and viral replicationmonitored by p24 ELISA assay at days 4-18 of culture. Data in FIG. 5 arefrom day 11. Three independent PBMC donors (all CCR5 wt/wt) gave similarresults as shown here, but other donors showed less enhancement of 241replication, or even partial inhibition (See FIG. 4 where AOP-RANTEStreatment of isolated CD4 T cells in vitro inhibits R5 viruses but canenhance R5X4 viruses in a CCR5-independent manner).

Example 17 V3 Sequences of the Macrophage-Tropic, CCR5 Using HIV-1Molecular Clone Used in These Experiments, and Virus Recovered fromHu-PBL-SCID Mice After AOP-RANTES Treatment

HIV-1 Clone 242 originated from B. Chesebro, and has the sequenceindicated in FIG. 3. The 242 virus pool used in these experiments has apoint mutation that resulted in a R to H change at position 21. Virusrecovered from all AOP-RANTES-treated mice as well as control miceretained the 242/H V3 sequence.

Examples 14-17 shows that AOP-RANTES has no effect on T-Tropic, X4 virusreplication. Further, AOP-RANTES may inhibit or enhance Dual-Tropic R5X4virus replication in vitro. Enhancement (when it occurs) is virus anddonor dependent, occurs at low doses of AOP-RANTES, and can occur incells from CCR5-null (del320 donors. AOP-RANTES is non-toxic in vivo andis able to delay but not prevent infection with an M-tropic R5 isolate.No selection for V3 mutation is observed (in contrast to the rapidselection for escape mutations induced by antibody treatment in thismodel).

Example 18 N-nonanoyl-RANTES (2-68) Inhibits HIV-1 Replication inhu-PBL-SCID Mice

Fifteen SCID mice were reconstituted with 20×10⁶ peripheral bloodmononuclear cells from an EBV-seronegative donor on day-14, usingmethods described in Mosier, Adv. Immunol. 63:79-125 (1996), Picchio, etal., J. Virol. 71:7124-7127 (1997), and Picchio, et al., J. Virol.72:2002-2009 (1998). The mice were divided into three groups of fivemice each. Group 1 received no additional treatment. Group 2 receivedimplantation of an Alzet 2001 mini-osmotic pump containing 500 μg ofn-nonanoyl-RANTES (2-68) (delivered at a rate of 1 μl per hour for aminimum of 200 hours). Group 3 received implantation of a similar Alzetpump containing 500 μg of BSA. The Alzet pumps were implantedsubcutaneously on day-1 (13 days after the SCID mice werereconstituted). Thus, on day-1, delivery of n-nonanoyl-RANTES (2-68)began for mice in Group 2. On day 0, all 15 mice were infected with 1000TCIDs of HIV-1 242, an isolate described in Chesebro, et al., supra, andSpeck, et al., supra. Mice in group 2 received an intraperitonealinjection of 1 mg n-nonanoyl-RANTES (2-68) one hour prior to virusinjection. N-nonanoyl-RANTES (2-68), was suspended in saline solutionand heated to 37° C.

Virus infection was monitored by plasmid HIV RNA levels, which weredetermined using the Roche Amplicor HIV Monitor assay, a quantitativePCR determination with a limit of detection of 200 copies/ml. Plasmasamples were obtained from all mice on day 7 (7 days after infectionwith HIV-1, 8 days after implantation of Alzet pumps in Groups 2 and 3),on day 14 (14 days after infection), and day 28 (28 days afterinfection). At the final time point, 3 of the 5 mice from each groupwere sacrificed for determination of the numbers of human CD3, CD4, CD8,and CD45-positive cells in two sites, the peritoneal cavity site of cellinjection, and local lymph nodes (“LN”) draining the peritoneal cavity.The frequency of human cell types was determined by staining withfluorochrome-labeled antibodies followed by fixation and flow cytometricanalysis, as described in Picchio, et al., J. Virol. 72:2002-2009 (1998)and Picchio, et al., J. Virol. 71:7124-7127 (1997).

Results for HIV RNA levels (in copies/ml plasma) are generally shown inFIG. 6 as log-transformed numbers. Log transformation is appropriatebecause viral replication is an exponential process, and smalldifferences in viral load (e.g., <half log₁₀) are insignificant. Cellrecovery is expressed as a percentage of total recovered cells, and CD4T cells are also expressed as a percentage of CD3 T cells, sinceCD4-positive cells are a subset of CD3-positive T cells. CD4 T cellrecovery experiments illustrate that n-nonanoyl-RANTES (2-68) can blockHIV-1 infection by a macrophage-tropic virus, 242 (now referred to as anR5 virus).

When virus RNA levels were undetectable, they are assigned the lowestvalue that could have been detected with the available volume of mouseplasma, e.g., 400 copies/ml. the number 400 in this context means 400copies or fewer, or undetectable by the Roche Amplicor assay. Recoveryof human cells at the end of the experiment has to be put into thecontext of what one would expect in the absence of HIV-1 infection.Historically, CD4 T cell counts account for 30-50% of human CD3 T cellsin the peritoneal cavity and 40-70% of total T cells in lymph nodes. Allmice in Group 2 (n-nonanoyl-RANTES (2-68) treated) had recovery ofCD4-positive T cells in the range expected for uninfected hu-PBL-SCIDmice. CD4 T cells accounted for 32-86% of total T cells in peritonealcavity in the 3 Group 2 mice, and from 52-68% of T cells in LN. Bycontrast, CD4 T cells in control groups 1 and 3 accounted for 8-12% oftotal T cells in peritoneal cavity, showing the CD4 T cell depletionpreviously documented in HIV-1 infected mice. Only one mouse (#124 ingroup 3) shows lower than expected levels of human cell and HIV-1 viralRNA.

Example 19 NNY-RANTES Blocks R5 Virus Entry In Vitro and in hu-PBL-SCIDMice Methods Generation of hu-PBL-SCID Mice

SCID mice were bred under specific pathogen flee conditions at ScrippsInstitute and tested for mouse IgM production at 8 weeks of age. Micewith <5 μg/ml of IgM were engrafted with PBMCs prepared fromEBV-seronegative donors from the Scripps General Clinical ResearchCenter pool. SCID mice were injected with 20×106 PBMC intraperitoneally,and checked for plasma levels of human IgG after 12-13 days. Micewith >100 μg/ml of human IgG were used for HIV-1 infection. Eachexperiment used mice generated from a single, different EBV-negativedonor.

HIV-1 Virus Pools

Infectious stocks of the 242 molecular clone were made by transfectingHEK 293 cells with a full length molecular clone provided by Dr. BruceChesebro. Virus recovered from the culture after 48 hours was used toinfect PBMC cultured for 4 days with PHA (2 μg/ml) and for 2 days withIL-2 (20 units/ml). Infectious virus was recovered after 7-10 days ofculture, and TCID of the virus determined by end-point titration. Micewere infected with 1000 TCID of virus. Sequencing results showed thatthe original 242 infectious stock differed from the published sequenceby having an H rather than R in position 21 of the V3 loop. This isshown in Table 1. This 242H variant was used for all the experimentsdepicted in FIG. 8. A second lot of 242 was prepared subsequently andshown to retain the original sequence. The original sequence was alsorecovered from one animal (NNY-R3 in FIG. 8B), which could have resultedfrom either mutation or selection of the original R sequence from avirus pool dominated by the 242H variant.

Table 1 shows the V3 envelope sequences of HIV-1 242 recovered fromhu-PBL-SCID mice treated with AOP- or NNY-RANTES. Sequences in B werederived from the two mice in FIG. 8B. The RSX4 241 isolate has an E to Qchange at position 24 (Chesebro, et al., supra) and retains the R atposition 21. 242H would thus require 2 amino acid changes to change celltropism and 242R only one amino acid change. TABLE 1 A.1       5         10        15        20        25        30      34 C TR P N N N T R R S I S I G P G R A F H T T E I I G D I R Q A H C stock —— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — all B.C T R P N N N T R R S I S I G P G R A F R T T E I I G D I R Q A H C — —— — — — — — — — — — — — — — — — — — R — — — — — — — — — — — — — NNY R3-1— — — — — — — — — — — — — — — — — — — — R — — — — — — — — — — — — — NNYR3-2 — — — — — — — — — — — — — — — — — — — — R — — — — — — — — — — — — —NNY R3-3 — — — — — — — — — — — — — — — — — — — — R — — — — — — — — — — —— — NNY R3-4 — — — — — — — — — — — — — — — — — — — — R — — — — — — — — —— — — — NNY R3-5 C T R P N N N T R R S I S I G P G R A F H T T E I I G DI R Q A H C — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —— — — — NNY R5-9 — — — — — — — — — — — — — — — — — — — — — — — — — — — —— — — — — — NNY R5-8 — — — — — — — — — — — — — — — — — — — — — — — — — —— — — — — — — — NNY R5-4 — — — — — — — — — — — — — — — — — — — — — — — —— — — — — — — — — — NNY R5-2 — — — — — — — — — — — — — — — — — — — — — —— — — — — — T — — — — — NNY R5-10

In Vitro Assays

PBMC were collected from normal blood by density centrifugation. CD4⁺Tcells were separated by depletion of other cell types by antibodytreatment and immunomagnetic bead separation. Whole PBMC or separatedCD4⁺ T cells were cultured at 5×104 cells per well in 96 well microtiterplates. Cells were activated with PHA and IL-2 for 34 days, the mediumreplaced with concentrations of AOP- or NNY-RANTES ranging from 100ng/ml to 1 pg/ml, cells incubated for 30 minutes at 37° C., and theninfected with 100 TCID of HIV-1 in the continued presence of modifiedRANTES. After overnight incubation, free virus was removed and freshmedium containing the original concentration of modified RANTES added.Culture medium was sampled on days 4, 7, and 10 after infection, and p24HIV capsid antigen measured by ELISA.

Administration of CCR5 Antagonists to Mice

AOP- or NNY-RANTES were dissolved in 0.9% saline at 2.5 mg/ml. Alzet2001 mini-osmotic pumps were loaded with 200-225 μl of compounds or BSAas a control. Pumps were surgically implanted subcutaneously underhalothane anesthesia between the scapulae, and the incision closed witha single wound clip. Pumps were observed for proper placement during thecourse of the experiment. A single intraperitoneal injection of 1 mg(0.4 ml) of either RANTES compounds or BSA was administered just priorto virus infection.

Virus Infection in Mice

Infection of hu-PBL-SCID mice with HIV-1 was determined by plasma HIV-1RNA levels measured by the quantitative Roche PCR assay (Amplicor HIVMonitor, Roche Molecular Systems, Somerville, N.J.). The limit ofdetection was 200-400 copies/ml depending on the plasma volumeavailable. Depletion of CD4⁺ T cells was measured by flow cytometry.Cells recovered from the peritoneal cavity or regional lymph nodes ofhu-PBL-SCID mice were stained with fluorescein- or phycoerythrin-labeledantibodies to human CD3, CD4, CD8, or CD45 and mouse H-2Kd (Pharmingen,San Diego, Calif.) and analyzed on a FACScan (Becton Dickinson, MountainView, Calif.) flow cytometer. CD4⁺ T cells are expressed as a percentageof total CD3+ cells.

RANTES Levels in Mice

Plasma from hu-PBL-SCID mice was analyzed for RANTES by ELISA (R & DSystems, Minneapolis, Minn.), using standard curves for either AOP- orNNY-RANTES. Plasma was diluted either 1:10 or 1:100 to bring the RANTESconcentration into the optimal sensitivity range of the assay.

V3 Envelope Sequences

RNA was extracted from mouse plasma using the Qiagen viral RNA kit(Qiagen, Valencia, Calif.). RNA was convened to cDNA by reversetranscriptase-PCR (RT-PCR). cDNA was amplified by nested PCR using thefollowing primers: outer V3 sense, CCAATTCCCATAGATTATTG; outer V3anti-sense, ATTACAGTAGAAAAATTCCCC; inner V3 sense,CAGTACAATGTACACATGGAATT; inner V3 anti-sense, AATTTCTGGGTCCCCTCCTGA. Thefinal 356 bp product was cloned using the TOPO TA Cloning Kit(Invitrogen, Carlsbad, Calif.), and the resulting product subject toautomated sequencing (ABI, Perkins-Elmer, Foster City, Calif.). Thefinal sequence encodes 54 amino acids 5′ of V3 and 50 amino acids 3′ ofV3. Although only the translated V3 sequence is shown in Table 1, theentire sequence was examined and there were no mutations outside of V3.

Results Inhibitory Activity of AOP- and NNY-RANTES In Vitro

The ability of AOP-RANTES and NNY-RANTES to inhibit R5 virus infection,including the R5 242 isolate of Chesebro, et al., supra, was confirmedby in vitro experiments (FIG. 7). The results show that NNY-RANTES andAOP-RANTES were both effective at inhibiting infection of activated PBMCwith the SF162 as well as the two variants of the 242 HIV-1 isolate (seeTable 1) and failed to inhibit infection with X4 isolates (data notshown). The CCR5 antagonists were able to reduce infection with the R5X4241 virus (Chesebro, et al., supra) only at higher concentrations, andshowed some enhancement of infection at lower concentrations (data notshown). NNY-RANTES was about three-fold more potent than AOP-RANTES atpreventing infection with SF162, but was not more potent than AOP-RANTESat inhibiting either the 242H or 242R variants. HIV-1 242R (with a Rrather than an H at position 21 of V3) was more resistant to inhibitionthan 242H with both CCR5 antagonists.

Activity of AOP- and NNY-RANTES in hu-PBL-SCID Mice

Three replicate experiments in hu-PBL-SCID mice were conducted toevaluate the in vivo efficacy of AOP- or NNY-RANTES. Because of theirrapid clearance from plasma, the CCR5 antagonists were administered atthe rate of 2.5 μg/hr by continuous infusion using subcutaneouslyimplanted osmotic pumps. In addition, a single dose of 1 mg (˜50 mg/kg)of each antagonist was injected just prior to virus infection. Serialplasma HIV RNA determinations were performed on the treated and controlhu-PBL-SCID mice following infection with HIV-1 242. In the experimentshown in FIG. 8A, mice were infused with AOP-RANTES or BSA as a control.Two of the four mice treated with AOP-RANTES had undetectable viral RNAlevels at the end of the 7 day infusion period, but virus levelsincreased in all mice once AOP-RANTES administration was halted. The oneanimal with viral RNA levels in the control range during treatment(AOP-R 4 in FIG. 8A) also had lower plasma levels of AOP-RANTES (SeeTable 2). AOP-RANTES was capable of reducing viral load but could notprevent HIV-1 infection despite plasma levels that were fully inhibitoryin vitro (see FIG. 7). The inhibitory capacity of NNY-RANTES was testedin the next two experiments using BSA as a control. The results of thefirst experiment are shown in FIG. 8B. Four of 5 hu-PBL-SCID infusedwith NNY-RANTES had undetectable viral RNA levels at the end of theinfusion period, and only 1 animal subsequently developed viremia (NNY-R3 in FIG. 8B). NNY-RANTES treatment was thus successful in preventing R5HIV-1 infection in 3 of 5 mice despite achieving lower plasmaconcentrations (Table 1) than AOP-RANTES. This experiment was repeatedusing a different human donor to generate hu-PBL-SCID mice. The results(FIG. 8C) were similar, with NNY-RANTES again preventing infection in 3of 5 mice.

Table 2 sets forth data as to the recovery of CD4⁺ human T cells,antagonist levels, and plasma HIV RNA in hu-PBL-SCID mice treated withCCR5 antagonists. Steady-state plasma levels of N-modified RANTES inhu-PBL-SCID mice are shown to be 3.63±0.8 ng/ml for AOP-RANTES and0.76±0.04 and 0.59±0.16 ng/ml for NNY-RANTES. TABLE 2 day 7 day 14N-RANTES day 14 HIV RNA Exp. Mouse ng/ml % CD4⁺ T cells log₁₀ copies/mlA AOP-R 1 4.51 17.1 3.98 AOP-R 2 4.77 29.2 6.07 AOP-R 3 4.03 27.7 3.86AOP-R 4 1.24 14.3 6.72 mean ± SE 3.63 ± 0.8  22.1 ± 3.7  5.16 ± 0.73 BSA1 <0.01 3.5 7.16 BSA 2 <0.01 11.9 6.06 BSA 3 <0.01 6.2 6.31 BSA 4 <0.014.4 6.12 mean ± SE — 6.5 ± 1.9 6.41 ± 0.25 B NNY-R 1 0.82 86.1 <2.30^(a)NNY-R 2 0.68 — <2.30 NNY-R 3 0.86 32.7 4.72 NNY-R 4 0.70 — <2.30 NNY-R 50.71 47.9 4.32 mean ± SE 0.76 ± 0.04 55.6 ± 15.9 — BSA 1 <0.01 11.7 6.49BSA 2 <0.01 12.3 6.36 BSA 3 <0.01 — 5.12 BSA 4 <0.01 — 6.34 mean ± SE —12.0 ± 0.3  6.08 ± 0.32 C NNY-R 1 0.49  nd^(b) <2.30 NNY-R 2 0.41 nd<2.30 NNY-R 3 1.23 nd <2.30 NNY-R 4 0.38 nd 6.02 NNY-R 5 0.43 nd 5.59mean ± SE 0.59 ± 0.16 — — BSA 1 <0.01 nd 6.49 BSA 2 <0.01 nd 6.36 BSA 3<0.01 nd 5.12 BSA-4 <0.01 nd 4.56 BSA-5 <0.01 nd 5.21 mean ± SE — — 4.72± 0.36^(a)indicates that the data was below the limit of detection of 200copies/ml^(b)not done

The relative survival of human CD4⁺ T lymphocytes in hu-PBL-SCID micetreated with CCR5 antagonists was also measured. Both AOP- andNNY-RANTES were able to slow the depletion of CD4⁺ T cells, even in micewhere HIV-1 infection was not prevented (See Table 2).

AOP- and NNY-RANTES Do Not Select for Co-Receptor Switch Variants

To determine if virus from hu-PBL-SCID mice that became infected despitetreatment with AOP-or NNY-RANTES was evading the antagonists by mutatingfrom CCR5 to CXCR4 co-receptor utilization, proviral DNA envelope geneswere amplified and the region surrounding the V3 loop was sequenced, acritical determinant of co-receptor usage (Cocchi, et al., Nature Med.2:1244-1247 (1996)). V3 sequences observed in the mice are shown inTable 1. In the first experiment (FIG. 8A), all mice treated withAOP-RANTES had the same sequence as the starting 242 virus isolate(which was found to contain an H in place of the published R at position21, a change that had occurred prior to the initiation of theseexperiments). In the second experiment (FIG. 8B), HIV-1 recovered fromthe two mice that became infected despite treatment with NNY-RANTESdiffered in the V3 sequence. One mouse had the sequence of the starting242 isolate (except for one clone with a replacement mutation atposition 29), while the other mouse showed a reversion of the H atposition 21 to the R present in the original molecular clone. Thepresence of H or R at position 21 in these isolates did not impact CCR5usage but did impact susceptibility to NNY-RANTES (FIG. 7B). Theseresults show that although sequence variation was occurring and theremay have been selection for sequence variants that were less sensitiveto NNY-RANTES inhibition, there was not rapid selection for HIV-1variants that used alternative co-receptors for viral entry.

Example 19 establishes that NNY-RANTES is more effective than AOP-RANTESin preventing HIV-1 infection, and that neither antagonist selected forviruses capable of utilizing other co-receptors for virus entry. Theseresults show that it is possible to block HIV-1 infection withN-terminally modified RANTES compounds in vivo. Inhibition of virusinfection occurred with plasma levels of 0.4-0.9 ng/ml of NNY-RANTES and4-5 ng/ml of AOP-RANTES during continuous administration of theantagonists, levels that are lower than the average concentration (˜20ng/ml) of native RANTES in human plasma (Weiss, et al., J. Infect. Dis.176:1621-1624 (1997)). There has been one previous report of a chemokinereceptor antagonist (AMD3100) that displayed efficacy against X4 HIV-1infection in mice, albeit at higher concentrations (Datema, et al.,Antimicrob. Agents Chemother. 40:750-754 (1996)), but this is the firstreport of antiviral activity of a CCR5 antagonist in vivo.

NNY-RANTES is as effective as a potent neutralizing antibody atpreventing HIV-1 infection of hu-PBL-SCID mice (Parren, et al., AIDS9:1-6 (1995) and Gauduin, et al., Nat. Med. 3:1389-1393 (1997)). Micethat were not protected from infection had lower vital RNA levels andhigher CD4⁺T cell counts than controls, suggesting that CCR5 antagonistsmay be useful in treating established infection.

These results also suggest that native RANTES concentrations in plasmaare generally too low to be a common explanation for virus resistance inexposed, uninfected individuals (Paxton, et al., Virology 244:66-73(1998)). The in vitro inhibitory concentrations of natural CCR5 ligandsare found to be in the 10-100 ng/ml range (Paxton, et al., supra andTrkola, et al., J. Virol. 72:396-404 (1998)), which is 10-100 foldhigher than inhibitory concentrations for AOP- and NNY-RANTES in vitro(Simmons, et al., supra) and in vivo (data in this Example). The normalplasma RANTES levels in most normal individuals are likely to be too lowto block virus infection, although some exposed, uninfected individualshave CD4⁺ T cells with high enough RANTES secretion to potentiallyprovide local protection (Paxton, et al., supra).

The absence of co-receptor switch variants following either AOP- orNNY-RANTES administration is consistent with the slow rate ofdevelopment of X4 viruses in infected humans The absence of co-receptorswitch variants following either AOP- or NNY-RANTES administration isconsistent with the slow rate of development of X4 viruses in infectedhumans (Schuitemaker, et al., J. Virol. 65:356-363 (1991); Tersmette, etal., Lancet 1:983-985 (1989); and Connor, et al., J. Virol. 67:1772-1777(1993)). Few mutations are required to change co-receptor usage (Speck,et al., J. Virol. 71:7136-7139 (1997)), suggesting that there must besignificant biological barriers to switching from the R5 to the X4 virusphenotype. In addition, therapies that target cellular rather than viralproteins are less likely to select for escape mutations. Therapies withmultiple chemokine receptor antagonists may also reduce the chance forescape mutations. It appears that one virus (242R, FIG. 7) with reducedsensitivity to NNY-RANTES emerged during treatment, so mutations thatalter sensitivity to CCR5 antagonists without changing co-receptor usagemay be anticipated. Nonetheless, these would be less troubling thanselection for more pathogenic X4 variants. The present in vivo resultsthus support the continuing development of co-receptor antagonists asviable candidates for the therapy of HIV-1 infection (Cairns, et al.,Nature Med. 4:563-568 (1998)).

Example 20 Capsule Formulation

This example illustrates the preparation of a representativepharmaceutical formulation for oral administration. Ingredients Quantityper capsule (mg) Active ingredient 200 lactose, spray-dried 148magnesium stearate 2The above ingredients are mixed and introduced into a hard-shell gelatincapsule.

Example 21 Tablet Formulation

This example illustrates the preparation of another representativepharmaceutical formulation for oral administration. A tablet for oraladministration is prepared having the following composition: IngredientsQuantity (mg/tablet) Active ingredient 400 corn starch 50 lactose 145magnesium stearate 5The above ingredients are mixed intimately and pressed into singlescored tablets.

Example 22 Oral Formulation

This example illustrates the preparation of a representative suspensionfor oral administration. Ingredients Quantity Active ingredient 1.0 gfumaric acid 0.5 g sodium chloride 2.0 g methyl paraben 0.1 g granulatedsugar 25.5 g sorbitol (70% solution) 12.85 g Veegum K (Vanderbilt Co.)1.0 g flavoring 0.035 mL colorings 0.5 mg distilled water q.s. to 100 mL

Example 23 Injectable Formulation

An injectable preparation buffered to a suitable pH is prepared havingthe following composition: Ingredients Quantity Active ingredient 0.2 gSodium Acetate Buffer Soln (0.4 M) 2.0 ml HCl (1N) or NaOH (1N) q.s. tosuitable pH water (distilled, sterile) q.s. to 20 ml

Example 24 Topical Formulation

This example illustrates the preparation of a representativepharmaceutical formulation for topical application. Ingredients Quantity(g) Active ingredient 0.2-10 Span 60 2 Tween 60 2 Mineral oil 5Petrolatum 10 Methyl paraben 0.15 Propyl paraben 0.05 BHA (butylatedhydroxy anisole) 0.01 Water q.s. to 100All of the above ingredients, except water, are combined and heated to60-70° C. with stirring. A sufficient quantity of water at 60° C. isthen added with vigorous stirring to emulsify the ingredients, and waterthen added q.s. to 100 g.

Example 25 Suppository Formulation

A suppository totaling 2.5 grams is prepared having the followingcomposition: Ingredients Quantity Active ingredient 500 mg witepsolH-15* q.s. to 2.5 g(*triglycerides of saturated vegetable fatty acid; a product of HULS,Inc., New Jersey).

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1: A compound of the formula: R¹-RANTES (2-68) where R¹ is CH₃—(CH₂)_(n)—X—; in which X is —C(O)—NH—CH₂—C(O)—, —NHCH₂—C(O)—, —ONH—CH₂—C(O)—, —OCH₂—CH₂—C(O)—, —CH═CH—C(O)—, —C(O)—, or a covalent bond; and n is an integer of 4-8; and in which RANTES (2-68) is a polypeptide having the sequence: (SEQ ID NO:2) PYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S

or is a polypeptide having a variant of said sequence, the variant sequence having at least 80% sequence homology with said sequence; wherein the compound further comprises one or more grafted organic chain-like molecules; or a pharmaceutically acceptable salt of the compound. 2: The compound of claim 1, wherein n is 4 and X is —C(O)—NH—CH₂—C(O). 3: The compound of claim 1, wherein n is 5 and X is —NH—CH₂—C(O)—. 4: The compound of claim 1, wherein n is 7 and X is —C(O)—. 5: The compound of claim 1, wherein n is 8 and X is a covalent bond. 6: The compound of claim 1, wherein n is 4 and X is —ONH—CH₂—C(O)—. 7: The compound of claim 1, wherein n is 5 and X is —CH═CH—C(O)—. 8: The compound of claim 1, wherein n is 4 and X is —OCH₂—CH₂—C(O)—. 9: The compound of claim 1, wherein said compound inhibits HIV-1 R % virus infection of PBMCs in vitro. 10: The compound of claim 1, wherein said compound binds to the RANTES CCR5 receptor. 11: The compound of claim 1, wherein RANTES (2-68) is a polypeptide having the sequence (SEQ ID NO:2) PYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S.

12: The compound of claim 1, wherein RANTES (2-68) is a polypeptide having the sequence (SEQ ID NO:2) PYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S

or a variant of said sequence having from 1 to 20 single amino acid deletions, insertions or substitutions. 13: The compound of claim 1, wherein one or more of said grafted organic chain like molecules are PEG-based chains. 14: The compound of claim 1, wherein one or more of said grafted organic chain like molecules is PEG. 15: The compound of claim 1, wherein said grafted organic chain-like molecules are grafted at the C-terminus. 16: A method of treating a disease state in a mammal that is alleviated by treatment with a RANTES inhibitor, which method comprises administering to the mammal in need of such treatment a therapeutically effective amount of the compound of claim
 1. 17: The method of claim 16, wherein the disease state is an inflammatory disease. 18: The method of claim 17, wherein the inflammatory disease is asthma, allergic rhinitis, atopic dermatitis, atheroma, atherosclerosis, or rheumatoid arthritis. 19: The method of claim 9, wherein the disease state is HIV. 20: A composition which comprises of a compound of claim 1 in admixture with one or more pharmaceutically acceptable excipients. 